Structure of Citrus Pectine

Structure of Citrus PectinMerve Kaya

supervisor: Dr. Marie-Christine Ralet INRA Nantes, France

jury members: Prof. Marc Hendrickx KU Leuven, Belgium

Prof. Jørn Dalgaard Mikkelsen Technical University of Copenhagen, Denmark

Prof. Peter A. Williams Glyndwr University Wrexham, United Kingdom

Prof. Phillippe Delavault University of Nantes, France

July 9, 2015Nantes, France

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Outline

Brief overview of pectin

Objectives

Materials & Methods

Strategy

Results

I. Characterisation of citrus pectin samples extracted under different conditions

II. Characterisation of isolated pectic domains

Summary

Perspectives

Plant cells are surounded by rigid cell walls

• cellulose

• hemicelluloses

• pectin

• mainly pectin

• mainly lignin (polyphenolic)

polysaccharides make up the major part of the primary cell wallsAlbersheim et al., 2010

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PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY

Why studying plant cell walls?

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food ingredientpharmacyproduct

industrial chemical

woodtechnology textile

dietary benefits animal feed


Pectin is a gelling, emulsifying, and thickening agent

• high sugar jam

• low sugar jam

• confectionery jellies

• yoghurt fruit preparations

• acidified dairy beverages

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CP Kelco, WallTraC training 2015

Commercial pectin is mainly derived from citrus peel

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• commercial availability

• pectin quality

• pectin yield

high production of citrus in the USA, Mexico, Brazil, China, and Spain


Orange production represent 60% of the world citrus production

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2010-world citrus production by fruit type

Turner & Burri, 2013

orange juice industry

• mostly preferred ones

≠pectin structureand functionality

grapefruit and pomelos

lemons and limes

oranges

tangerines, mandarins, clementines

other


Pectin extraction

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chopping and washing

after drying

transportation

• alcohol precipitation

• acidified hot water(nitric acid)

extraction filtration purification


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xylogalacturonan (XGA)

arabinogalactan II

arabinans

homogalacturonan (HG)

rhamnogalacturonan II (RGII)

Pectin comprises structurally distinct pectic domains α–D-GalpA

α-L-Rhap

α-L-Araf

β–D-Galp

α-D-Xylp

β–D-DHAp

β-L-Araf

α-D-KDOp

β-D-Apif

β-L-Rhap

β–L-Galp

β-D-GlcpA

α–L-Galp

β-D-GalpA

β-D-Fucp

α-L-Arap

β-L-AcefA

Ropartz, 2015

methyl esterification

acetyl esterification

rhamnogalacturonan I (RGI)

two main domains

arabinogalactan I


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homogalacturonan

HG is the predominant domain

• exclusively composed of α–(1, 4)-linked D-GalpA

• unbranched polymer

• chain length is approximately 100 GalA residues

Voragen et al., 1995; Thibault et al., 1993; Hellin et al., 2005

α–D-GalpA


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homogalacturonan

HG is the predominant domain

• exclusively composed of α–(1, 4)-linked D-GalpA

• unbranched polymer

• chain length is approximately 100 GalA residues

• GalA residues are partly methyl/ acetyl esterified

Degree of methyl esterification (DM)

• DM > 50 %, High methyl-esterified pectin (HM)

• DM < 50 %, Low methyl-esterified pectin (LM)

Voragen et al., 1995; Thibault et al., 1993; Hellin et al., 2005

α–D-GalpA




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Gelling mechanism

Voragen et al., 1995; Powell et al., 1982; Oakenful & Scott, 1984; Rolin, 2002

Calcium• LM pectin gel according to ‘’egg box’’ model

• 7-20 non-esterified GalA residues are required

• form gel at pH 3-6, sugar is not necessary

• HM pectin gel due to hydrogen bonds and

hydrophobic interaction between methylated groups

• form gel at pH 2-3.8 and 60% sugar

homogalacturonan

α–D-GalpA



• alternating GalA and Rha residues

RGI backbone

Lau et al., 1985

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α–D-GalpA

α-L-Rhap

acetyl esterification on GalA residues



RGI side chains

Lau et al., 1985; Albersheim et al., 1996; Ridley et al., 2001

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arabinogalactan II

arabinogalactan I

arabinans

• mainly Gal and Ara residues are attached to Rha

• 20-80% of Rha branched with neutral sugar side chains

α–D-GalpA

α-L-Rhap

α-L-Araf

β–D-Galp

• (1, 5)- α-L-Araf backbone

• branched by α-L-Araf units

• (1, 4)-β–D-Galp backbone

• short side chains of (1, 5)- α-L-Araf

• (1, 3)-β–D-Galp backbone

• side chains of (1, 6)-β–D-Galp

• Ara residues can be attached

single β–D-Galp


Smooth and Hairy Regions

Rhamnogalacturonan-I Backbone

Homogalacturonan

Neutral sugar side chains

Rhamnogalacturonan I backbone

Model 1

Model 2

(de Vries, 1981; Schols and Voragen, 1996)

(Vincken et al., 2003).

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How these pectic domains are connected to each other?


• GalA & neutral sugar content

• molecular weight and intrinsic viscositygelling strength

stabilising power

Structure- function relationship

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• HG length

• HG/ RGI proportion


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Ara-containing side chains important for gel strength of Ca2+-pectin gels

enzymatically modified pectin

(Ara- containing side chain degrading enzymes)

• significant reduction in Ara content

Total NSGalAraRha

WSP: water soluble carrot pectin DBr: debranchedDEP: de-esterified pectin NS: Ara+Gal+Rha

WSP WSPDBr DEP DEPDBr

Ngouemazong et al., 2012

• ‘’weak’’ gel behaviour

(induced entanglement of the polymer)


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• to apprehend possible structural and macromolecular variation in

extracted-pectin samples related to citrus source

• to determine the effect of extraction conditions (pH and extraction

agent) on pectin and pectic sub domain characteristics


plant source

acid type

nitric acid

oxalic acid

pH

mild

harsh

MN: mild nitric acid MO: mild oxalic acid HN: harsh nitric acid HO: harsh oxalic acid

extractionconditions

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plant source

acid type

nitric acid

oxalic acid

pH

mild

harsh

MN: mild nitric acid MO: mild oxalic acid HN: harsh nitric acid HO: harsh oxalic acid

extractionconditions

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Oxalic acid is a chelating agent

• oxalic acid: isolation of cation-based (mostly Ca2+) cross-linking


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2. isolated pectic domains

acidic means hot alkalirhamnogalacturonan I-RGI

homogalacturonan-HG

hot alkali1. characterisation of

extracted-pectin samples


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1. extracted pectin samples

• yield• sugar composition

• molecular weight & intrinsic viscosity


Extraction yields

harshnitric acid

harshoxalic acid

mild oxalic acid

mild nitric acid

mg extract/ g dry peel

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pH of extraction

orange lemon

lime grapefruit


Extraction yields

the lowest yield with orange

mg extract/ g dry peel

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pH of extraction

orange lemon

lime grapefruit


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1. extracted pectin samples• yield

• sugar composition• molecular weight & intrinsic viscosity


Arabinose + Galactose

Rhamnose

RGI “decoration”

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arabinogalactan II

arabinogalactan I

arabinansRha: branching point

Ara and Gal: major sugars


• nitric acid: trim side chains

(especially Ara-containing ones)

harsh nitric acid

harshoxalic acid

mild oxalic acidmild

nitric acid


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Ara + Gal

Rha

pH of extraction

orange lemon

lime grapefruit


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harsh nitric acid

harshoxalic acid

mild oxalic acidmild

nitric acid


pH of extraction

orange lemon

lime grapefruit

• nitric acid: trim side chains

(especially Ara-containing ones)

• oxalic acid: conserved

abundant side chains

Ara + Gal

Rha


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pH of extraction

• grapefruit has low amount

of neutral sugar

• İt indicates shorter/fewer

side chains

orange lemon

lime grapefruit Ara + Gal

Rha


Galacturonic acid

Rhamnose

HG/ RGI ratio

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GalA

Rha


pH of extraction

HG/ RGI ratio

nitric acid isolated RGI rich

pectin

oxalic acid isolated HG rich

pectin

harsh nitric acid

mild nitric acid

harshoxalic acid

mildoxalic acid

GalA/ Rha

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orange lemon

lime grapefruit


pH of extraction

HG/ RGI ratio

GalA/ Rha

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orange and grapefruit were

rich in RGI

orange lemon

lime grapefruit


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1. extracted pectin samples• yield

• sugar composition

• molecular weight & intrinsic viscosity


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What is IV [η] (Intrinsic Viscosity)?

• HG is a rigid polymer

• RGI is more flexible

• IV is lower

the molecule is compact,

occupying a relatively small volume


• nitric acid: hydrolysis of neutral

sugar side chains, breakdown of RGI

backbone, and possible breakdown of

HG domains

harshnitric acid

mildnitric acid

mild oxalic acidharsh

oxalic acid(dL/ g)

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Intrinsic viscosity

pH of extraction

orange lemon

lime grapefruit


pH of extraction

Intrinsic viscosity

• nitric acid: hydrolysis of neutral

sugar side chains, breakdown of RGI

backbone, and possible breakdown of

HG domains

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harshnitric acid

mildnitric acid

mild oxalic acidharsh

oxalic acid(dL/ g)

orange lemon

lime grapefruit

• oxalic acid: dissolution of better-

conserved pectin structure


orange and grapefruit: lower IV

Rha-rich samples are more flexible

orange lemon

lime grapefruit

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pH of extraction

(dL/ g)

Intrinsic viscosity

Axelos & Thibault, 1991; Ralet et al., 2008


• oxalic acid extracted pectins rich in

HG & RGI stretches with conserved side chains

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Homogalacturonan




• nitric acid extracted pectins exhibited

lower Mw & IV & RGI stretches with few and/or short side chains

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Homogalacturonan




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acidic means

homogalacturonan-HG

1. characterisation of


yield

sugar composition

macromolecular characteristics


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HG/ RGI ratio


REDACTED

HG/ RGI ratios of pectin samples

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REDACTED

Degree of polymerization of HG domains

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REDACTED

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acidic means hot alkalirhamnogalacturonan I-RGI

homogalacturonan-HG

hot alkali1. characterisation of


yield

sugar composition

macromolecular characteristics

+ purification by

chromatography


Sugar analyses of purified RGI regions of lemon

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REDACTED


HGs :

• REDACTED

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• REDACTEDKey findings


• GalA & neutral sugar content

• molecular weight and intrinsic viscosity

• HG length

• HG/ RGI proportion

important for pectin applicability

gelling strength

stabilising power

Impact of pectin structural variances on rheological performances?

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Acknowledgment

WallTraC fellows & PIs

Friends…

INRA colleagues

CP Kelco colleagues

Family..

Dr. Marie Christine Ralet

PVPP Team

Special thanks: • Marie-Jeanne Crepeau• Jacqueline Viqouroux• Susanne Sorensen• Antonio Sousa

Acknowledgment

This presentation reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein.

More information about the WallTraC project at www.walltrac-itn.eu.

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7 2007-2013) under Grant Agreement n°263916.

http://www.walltrac-itn.eu/

Science

Structure of Citrus Pectine